1. Field of the Invention
The present invention relates to microfluidic devices for capturing single cells, that can capture cells contained in a sample at one-cell level, methods for separating and capturing cells contained in a sample at one-cell level using a microfluidic device, and methods for quantitatively analyzing gene expression of a single cell utilizing a microfluidic device.
2. Description of the Related Art
Stochasticity in gene expression, protein or metabolite levels contributes to cell-cell variations, the analysis of which could lead to a better understanding of cellular processes and drug responses. Conventional technologies are limited in their throughput, resolution (in space, time, and tracking individual cells instead of population average) and the ability to control cellular environment. A few microfluidic tools have been developed to trap and image cells; however, in most designs presently available, there is a disadvantageous compromise among loading efficiency, speed, the ability to trap single cells, and density or number of trapped cells.
Stochastic effects in gene expression and transcription events in mammalian cells lead to large variations in messenger ribonucleic acid (RNA) copy numbers, causing cell-to-cell variability in genetically identical cells. A current view is that noise arising from stochastic fluctuations plays an essential role in key cellular activities. For example, clonal populations of mouse multipotent progenitor cells or cancer cells have differential fate outcome in response to the same uniform stimulus because of heterogeneities in the dynamics of regulatory proteins, in the expression level of basal signaling proteins, and/or states of proteins regulating apoptosis. Tracking single cell dynamic response is therefore necessary to monitor stochastic fluctuations among cell populations.
At present, such experiments can be technically challenging if the cells of interests are non-adherent, if stimuli need to be delivered, and/or if studies on long time scales are desired. Flow cytometry is often the technique of choice to measure heterogeneity of suspension cell populations, as it can provide high-throughput and can distinguish subpopulations of cells. However, this technology is capable of neither monitoring temporal changes within the same cell, nor distinguishing population from noise due to temporal fluctuation within one cell. Quantitative time lapse microscopy is often required for these measurements, but it presents additional challenges, such as relatively low throughput and movement of the target cells during imaging. It is particularly challenging to image suspension cells. Although one could use adhesion to a solid surface by use of an artificial membrane and receptor binding, this may alter the biological behavior of the cells.
In an attempt to overcome limitations of traditional real-time microscopy, microfluidics has been proposed to allow for increased throughput, control of cell location and extracellular conditions. Various microfluidic techniques have been developed to capture cells, retain them in a specific location, and control the environment surrounding them. Although some of these techniques are quite powerful, even these methods have a limited throughput because the cell traps are spaced sparsely enough such that per view only a small number of cells are captured, and some are difficult to implement, or have side effects or other limitations.
For example, active single-cell capture mechanisms use valves to control flow or dielectric forces with dielectrophoresis (DEP) or optical tweezers to control the location of cells in various environments. Yet the use of dielectric forces on living cells limits cell viability due to buffer cytotoxicity and heat damage.
Passive capturing mechanisms have also been proposed using gravity or fluid flow to direct cells into traps. Most microwell arrays rely on gravity to capture cells. Careful design of the microwells enables up to 70% single cell capture in densely packed wells, but once trapped, exposure to varying chemical solutions and manipulation of the cells are limited because the cells are not actively held in the wells. Flow by diverting streamlines towards traps can also be used to transport and dock cells at specific locations. Once a trap contains cells, fluid towards the trap is significantly reduced, and therefore incoming cells will be diverted to the next empty trap. Optimization of trap dimensions, location and spacing has been performed to increase capture efficiency or single cell trapping. However, in most designs to date, there is a compromise between cell trap density per area and single cell capture efficiency.
What is needed, therefore, is a microfluidic high-density single cell capture, stimulation, and imaging platform that can passively trap thousands of cells relatively quickly with a relatively high single-cell loading efficiency. It is to these needs that the present invention is primarily directed.